Optoelectronic sensor

ABSTRACT

A device for detecting the presence of an antigen including (1) a cell having antibodies which are expressed on the surface of the cell and are specific for the antigen to be detected, where binding of the antigen to the antibodies results in an increase in calcium concentration in the cytosol of the cell, the cell further having a emitter molecule which, in response to the increased calcium concentration in the cytosol, emits a photon; (2) a liquid medium for receiving the antigen and in which the cell is immersed; and (3) an optical detector arranged for receiving the photon emitted from the cell.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/471,275 filed Jun. 19, 2006, which is a continuation of U.S.application Ser. No. 10/910,554 filed Aug. 3, 2004, now U.S. Pat. No.7,090,988, which is a continuation of U.S. application Ser. No.09/848,811 filed May 4, 2001, now U.S. Pat. No. 6,800,448, which is acontinuation of U.S. application Ser. No. 09/169,196 filed Oct. 9, 1998,now U.S. Pat. No. 6,248,542, which is a continuation-in-part of U.S.application Ser. No. 08/987,410 filed Dec. 9, 1997, now U.S. Pat. No.6,087,114. The entire teachings of the above applications areincorporated herein by reference.

GOVERNMENT SUPPORT

The invention was supported, in whole or in part, by Contract NumberF19628-95-C-0002 awarded by the U.S. Air Force. The government hascertain rights in the invention.

BACKGROUND OF THE INVENTION

The need for small, fast, and sensitive detectors of biological agentswhich are able to continuously monitor an environment for extendedperiods of time is underscored by the proliferation of biological andchemical weapons, the poor man's nuclear weapon. Under battlefieldconditions, a useful detector would rapidly alert a soldier when aspecific biological or chemical agent is detected so thatcountermeasures can quickly be implemented.

Such detectors would be useful in non-military applications as well.Rapid detection of antibiotic-resistant bacteria in a patient would helpclinicians select a more effective therapeutic regimen. Continuousmonitoring of a city's drinking water supply would provide early warningof potential pathogens, giving public works officials more time tomanage the potential health risks to the public. In addition, the use ofthese detectors in meat and poultry inspections would be a significantimprovement over the current “poke-and smell” procedure.

All vertebrates acquire a specific immune response to a foreign agent(antigen) in part by generating an immense diversity of antibodymolecules. Antibody molecules bind to antigen with high specificity,e.g., they can differentially bind to two closely related strains ofbacteria or viruses.

Antibodies are produced by B cells, a crucial component of the immunesystem. An antigen can activate a B cell by binding to antibodies on itssurface, leading to a cascade of intracellular biochemical reactionswhich causes a calcium ion influx into the cytosol of the B cell.

For a review of antibody structure and function and B cell activation,see Paul, editor, Fundamental Immunology, 3^(rd) ed., Raven Press, NewYork (1993).

SUMMARY OF THE INVENTION

This invention relates to a device for detecting an antigen. The deviceincludes a liquid medium containing cells (e.g., a B cell or fibroblast)and an optical detector, the liquid medium receiving the antigen. Eachof the cells has antibodies (e.g., chimeric or single chain antibodies)which are expressed on its surface and are specific for the antigen tobe detected. Binding of the antigen to the antibodies results in anincrease in calcium concentration. The cells also contain emittermolecules (e.g., aequorin or indo-1) in their cytosol which emit photonsin response to the increased calcium concentration in the cytosol. Thedetector can be separated from the medium containing the cells by acovering (e.g., glass) that is transparent to the photons. Such acovering can serve to support the medium, protect a fragile surface ofthe detector, or be used as a lens. The optical detector, e.g., acharge-coupled device (CCD) is able to detect the photons emitted fromthe cells in response to the increase calcium concentration and indicateto the user that the antigen to be detected is present. Other opticaldetectors which can be used in the device include a photomultiplier tubeor a photodiode. In some embodiments, the optical detector is able todistinguish individual cells.

The device can be contained within a housing made from, for example,aluminum, plastic, or stainless steel. Such a housing can preventcontamination of the device with extraneous organisms. The housing caninclude two halves attached to each other on one side of the housing bya hinge joint. In applications where an airborne antigen is to bedetected, the housing can contain one or more openings for the antigento pass into the device. Such an opening can be screened by anantigen-permeable barrier such as a metal mesh or a membrane.

The sample containing the antigen can pass through a filter before theantigen contacts the cells. Suitable filters include passive filters(e.g., filter with determined pore sizes, affinity columns orimmunofilters) and active filters (e.g., fluorescence-activated sorters,active size sorters, or microfluidic systems).

The invention also features a method for detecting the presence of anantigen, which includes providing a sample (e.g., a volume of air)suspected of containing the antigen; introducing the sample into adevice containing cells immersed in a medium; and monitoring photonemission as an indication of whether the antigen is present. The cellsused in this method are described above.

Further, the invention includes a cell containing an emitter moleculeand having antibodies on its surface, so as to be useful in the devicesand methods of the invention.

Other features or advantages of the present invention will be apparentfrom the following drawings, detailed description, and the claims. Anypublications cited in this disclosure are hereby incorporated byreference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is perspective view of a device for detecting an antigen in itsopen position.

FIG. 2 is a top view of the device in FIG. 1 with the upper half of thehousing removed.

FIG. 3 is a cross-sectional view of the device taken along sectionalline A-A of FIG. 2.

DETAILED DESCRIPTION OF THE INVENTION

The invention is based on the discovery that cells having antibodies onits surface and containing a compound that emits a photon upon externalstimulation by an antigen can be used in an optoelectronic sensor. Thebiological aspect of the invention are discussed below.

I. Cells

The cell which has surface-bound antibodies can be either prokaryotic oreukaryotic. Upon binding of antigen to the antibodies, the cellmobilizes calcium ions into the cytosol. An example of a cell useful inthe device and methods of the invention is a B cell (i.e., a B cell froma cold or warm-blooded vertebrate having a bony jaw) which can begenetically engineered to express one or more surface-bound monoclonalantibodies. It also can be produced by, for example, immunizing ananimal with the antigen to be detected and harvesting the B cell fromthe immunized animal. The harvested B cells can be further immortalizedand screened for production of a surface monoclonal antibody specificfor the antigen to be detected.

Alternatively, the cell can be a fibroblast, which offers the advantagethat the cell can be adhered to a substrate of a device of theinvention. However, fibroblasts do not contain the signal transductionmachinery necessary to transfer a signal from the cytoplasmic portion ofa surface antibody to calcium stores in the cell. To overcome thisproblem, a chimeric surface antibody can be expressed in the fibroblast.This chimeric antibody contains a cytoplasmic amino acid sequencederived from a polypeptide (e.g., a fibroblast growth factor receptor)that can transduce a signal from the inner surface of the plasmamembrane of the fibroblast to intracellular calcium stores. Thus, whenan antigen binds to the extracellular portion of the chimeric antibodyto cause antibody aggregation on the surface, calcium mobilization isinduced. A similar strategy using chimeric antibodies can be employedfor any other cell type which is not a B cell, so that the cell issuitable for use in the devices and methods of the invention.

Growth of the cell can be controlled by any means well known in the art,including providing anti-mitotic drugs (e.g., α-amanitin) or growthfactors (e.g., fetal bovine serum) in the medium. Alternatively, cellscan be genetically engineered to grow at a determined rate. As discussedabove, any cell can be used as long as binding of the antigen to theantibodies on the surface of the cell leads to an increase in calciumconcentration in the cytosol. In fact, the cell can be a non-living,manufactured unit as long as it satisfies the above requirement.

II. Antibodies

An antibody which specifically binds to the antigen to be detected is amolecule which binds to the antigen or a epitope of the antigen, butdoes not substantially bind other antigens or epitopes in the sample.Such antibodies can be chimeric (i.e., contain non-antibody amino acidsequences) or single chain (i.e., the complementarity determining regionof the antibody is formed by one continuous polypeptide sequence).

Polyclonal cells expressing antibodies can be prepared by immunizing asuitable animal with the antigen to be detected. The cells producingantibody molecules directed against the antigen can be isolated from theanimal (e.g., from the blood) and further purified by well-knowntechniques, such as panning against an antigen-coated petri dish.

Alternatively, surface antibody-producing cells can be obtained from theanimal and used to prepare a monoclonal population of cells producingsurface antibodies by standard techniques, such as the hybridomatechnique originally described by Kohler et al., Nature 256:495-497(1975); Kozbor et al., Immunol Today 4:72 (1983); or Cole et al.,Monoclonal Antibodies and Cancer Therapy, Alan R. Liss Inc., pp. 77-96(1985). The technology for producing cells expressing monoclonalantibodies is well known (see e.g., Current Protocols in Immunology(1994) Coligan et al. (eds.) John Wiley & Sons, Inc., New York, N.Y.),with modifications necessary to select for surface antibodies ratherthan secreted antibodies.

Any of the many well known protocols used for fusing lymphocytes andimmortalized cell lines can be applied for the purpose of generating acell producing a surface monoclonal antibody (see. e.g., CurrentProtocols in Immunology, supra; Galfre et al., Nature 266:55052, 1977;Kenneth, in Monoclonal Antibodies: A new Dimension In BiologicalAnalyses, Plenum Publishing Corp., New York, N.Y., 1980; and Lerner,Yale J Biol Med 54:387-402 (1981). Moreover, the ordinarily skilledworker will appreciate that there are many variations of such methodswhich also would be useful.

As an alternative to preparing monoclonal cells, a nucleic acid encodinga monoclonal antibody can be identified and isolated by screening arecombinant combinatorial immunoglobulin library (e.g., an antibodyphage display library) with the antigen to thereby isolateimmunoglobulin library members that bind the antigen. Kits forgenerating and screening phage display libraries are commerciallyavailable (e.g., the Pharmacia Recombinant Phage Antibody System,Catalog No. 27-9400-01; and the Stratagene SurfZap™ Phage Display Kit,Catalog No. 240612). Additionally, examples of methods and reagentsparticularly amenable for use in generating and screening antibodydisplay library can be found in, for example, U.S. Pat. No. 5,223,409;PCT Publication No. WO 92/18619; PCT Publication No. WO 91/17271; PCTPublication WO 92/20791; PCT Publication No. WO 92/15679; PCTPublication WO 93/01288; PCT Publication No. WO 92/01047; PCTPublication WO 92/09690; PCT Publication No. WO 90/02809; Fuchs et al.,Bio/Technology 9:1370-1372 (1991); Hay et al., Hum Antibod Hybridomas3:81-85 (1992); Huse et al., Science 246:1275-1281 (1989); Griffiths etal., EMBO J 12:725-734 (1993).

After the desired member of the library is identified, the specificsequence can be cloned into any suitable nucleic acid expresser andtransfected into a cell such as a fibroblast. The expressor can alsoencode amino acids operably linked to the antibody sequence asappropriate for the cell which is to express the antibody. As discussedabove, the cytoplasmic transmembrane sequence of a fibroblast growthfactor receptor can be linked to a single-chain antibody specific forthe antigen to be detected, so that the cell immobilizes calcium whencontacted with the antigen. Although separate recombinant heavy chainsand light chains can be expressed in the fibroblasts to form thechimeric antibody, single chain antibodies also are suitable (see, e.g.,Bird et al., Trends Biotechnol 9:132-137, 1991; and Huston et al., IntRev Immunol 10:195-217, 1993).

III. Photon Emitter Molecules

A suitable emitter molecule is any molecule that will emit a photon inresponse to elevated cytosolic calcium concentrations, includingbioluminescent and fluorescent molecules. One emitter molecule, thebioluminescent aequorin protein, is described in Button, et al., CellCalcium 14:663-671 (1993); Shimomura et al., Cell Calcium 14:373-378(1993); and Shimomura, Nature 227:1356-1357 (1970). Aequorin generatesphotons by oxidizing coelenterazine, a small chemical molecule.Coelenterazine diffuses through cellular membranes, so coelenterazine oran analog thereof can be added to the culture medium surrounding thecells. Alternatively, genes encoding enzymes that make coelenterazinecan be introduced into the cells. In another embodiment, bioluminescentgreen fluorescent protein (GFP) can be used (see Chalfie, PhotochemPhotobiol 62:651-656 [1995]). In this embodiment, the cell cytosolcontains both GFP and aequorin. In response to elevated calcium in thecytosol, aequorin donates energy to GFP in an emissionless energytransfer process. GFP then emits the photon. Alternatively, the emittermolecule can be a calcium-sensitive fluorescent molecule (e.g., indo-1)which is illuminated by a wavelength of light suitable to inducefluorescence.

Aequorin, or any other emitter molecule, can be introduced into thecells by methods well known in the art. If the emitter molecule is aprotein (as is the case with aequorin), the cell can contain anexpression vector encoding the protein (i.e., a nucleic acid or viruswhich will produce the emitter molecule when introduced into a cell). Anexpression vector can exist extrachromosomally or integrated into thecell genome.

IV. Detection of One or More Antigens

The antigen can be introduced into the device passively. For example,airborne anthrax bacteria can enter the device through an opening viaair currents. Alternatively, an airborne antigen can be activelyintroduced into the device by e.g., a fan. The antigen to be detectedcan also reside in an aqueous medium. For example, a drop of the aqueoussample can be added to the medium containing the cells.

Instead of grouping cells in sectors with an array, the cells specificfor different antigens can be interspersed if the photon emitted from acell specific for one antigen is of a wavelength different from thephoton emitted from a cell specific for another antigen. In such asituation, the optical detector can differentially detect the photons ofdifferent wavelengths. For example, cells sensitive to differentantigens can contain different aequorin-like proteins and/or differentfluorescent proteins so that the different cells could be distinguishedon the basis of differing photon emission characteristics (e.g., peakwavelength, spectral width of emission, or decay rate). To allowidentification of a large number of antigens, signal multiplexing canalso be used. For example, the emission spectrum of a given type of cellcould have multiple identifiable peaks. Different fusion proteins (e.g.,aequorin and a fluorescent protein joined together by fusing their genesin frame) could be useful for this purpose.

In general, multivalent antigens (which contain multiple binding sitesfor a specific antibody) are required to cross-link surface antibodieson the cell in order to trigger calcium influx into the cytosol.However, monovalent antigens (which contain only one binding site for aspecific antibody) can also be detected by the device. Such antigens canbe detected if several monovalent antigens are linked together (bynoncovalent bonding, for example) as the antigens bind to the antibodieson the surface of the cell. Alternatively, the cell can be geneticallyengineered to expressed two or more antibodies which are specific to thesame antigen but bind to spatially separated epitopes of the antigen,thereby allowing a single antigen to bind to at least two antibodies onthe cell surface with different specificities.

Without further elaboration, it is believed that one skilled in the artcan, based on the above disclosure and the examples below, utilize thepresent invention to its fullest extent. The following examples are tobe construed as merely illustrative of how one skilled in the art canpractice the invention, and are not limitative of the remainder of thedisclosure in any way. Any publications cited in this disclosure arehereby incorporated by reference.

EXAMPLE 1 Optoelectronic Device with Housing

Referring to FIGS. 1-3, a device 1 for detecting multiple airborneantigens includes a housing 2 having an upper half 3 and a lower half 3′which together define an inner volume 18 (FIG. 3) of the device. Whenupper half 3 and lower half 3″ are together (closed position), they alsodefine an opening 4 through which the antigen to be detected enters thedevice 2. An antigen-permeable mesh 9 is positioned over the opening 4to prevent the ingress of large particles which could interfere withproper functioning of the inner components. The upper half 3 of thehousing 1 is attached to the lower half 3′ by hinge 5, allowing thedevice 1 to be opened in a direction as indicated by arrow 8. Lower half3′ has an open bottom to receive an optical detector, here a CCD 6,which is discussed in greater detail below.

The top view of the device 1 when the upper half 3 of the housing 2 isremoved is shown in FIG. 2. The device 1 includes an array 10 of sectorsA-P, each sector includes liquid medium 16 containing cells 12 immersedtherein. The liquid media 16 in the sectors are separated from eachother by walls 17 to prevent intermixing of cells in each sector. Thespecific cells provided within the medium of a sector can all be thesame type or may be specific to a particular antigen to be detected. Inthe latter case, the device 2 is capable of detecting a variety ofdifferent antigens. Although walls 17 are used as barriers in thisembodiment, they are not always necessary. For instance, when the mediumcontaining the cells are droplets of an aqueous solution, the dropletsremain within the sector by water surface tension. Alternatively, thebarriers can be edged grooves running between the sectors, in which casewater surface tension at the edge of the groove will keep the dropletsfrom intermixing. In another example, the cells are adhered to a surfaceand intermixing of the media in different sectors is not a problem.

Referring to FIG. 3, the optical detector 6 includes a detection surface13. The optical detector has a glass covering 14 which is in contactwith the liquid medium 16 containing cells 12. To increase photondetection efficiency, the device 1 includes a photon reflective layer 15located on the inner surface of upper half 3 of the housing 2.

When the antigen to be detected binds to the antibodies on the surfaceof cells 12, calcium ions move into the cytosol as described in Wilsonet al., J Exp Med 166:601-606 (1987). The increased cytosolic calciumconcentration causes an emitter molecule to emit a photon, which is readby the optical detector 6. In one embodiment, each cell will havemultiple copies of the emitter molecule and will emit multiple photonswhen activated. Optical detector 6 provides electrical signalsrepresentative of an image of array 10. The electrical signals areprocessed to indicate which of sectors of array 10 have detected thepresence of an antigen. For example, the signals are received by aprocessor 20 which is connected to indicator 22 (e.g., a light or soundemitting device) of the presence of one or more of the antigens. In oneembodiment, for example, indicator 22 includes lamps, each of which isassociated with one of the sectors of array 10 and provides anindication of the presence of the antigen associated with that sector.

The arrangement of the optical detector 6 with respect to the mediacontaining the cells can be varied. For example, the medium 16 couldfill inner volume 18 between the glass covering 14 and the reflectivelayer 15, and the opening 4 is covered by a membrane or porous filterinstead of mesh 9. In this case, the cells adhere to glass covering 14,rendering any walls 17 between sectors unnecessary. To facilitateadherence of the cells 12 to the glass covering 14, the glass can becoated with substances (e.g., poly-L-lysine or extracellular matrixproteins). In this variation of the device, an antigen in a liquid canbe detected as long as the liquid containing the antigen contacts themembrane and diffuses through medium 16 to the cells 12. Alternatively,the housing 2 can be opened by pivoting upper half 3 and a liquid samplecontaining the antigen to be detected can be dropped into the medium 16.

The liquid medium 16 can contain nutrients, dissolved gases, cytokines,antibiotics (to inhibit contamination), or any other substance necessaryfor maintenance of the cells. Furthermore, the liquid medium can containsubstances to aid the detection of calcium in activated cells (e.g.,coelenterazine substrate or analogues thereof to recharge spent aequorinmolecules or calcium-sensitive fluorescent substances). If necessary,the medium is maintained at a suitable temperature or pH.

EXAMPLE 2 Use of B Cells in an Optoelectronic Sensor

The ability of B cells to emit photons in response to an extracellularsignal was evaluated. K46J B cells were loaded with thecalcium-sensitive fluorescent dye indo-1, stimulated by contacting theloaded cells with anti-IgM antibodies, and monitored for fluorescence asfollows.

About five million cells were removed from their growth medium andresuspended in 1 ml Hank's Balanced Salt Solution (HBSS). One vial ofindo-1 was mixed with 113 μl of fetal bovine serum (FBS) and 25 μl ofpluronic F-127 detergent (20% in DMSO). The mixture was incubated atroom temperature for five minutes. The FBS was necessary to solubilizethe indo-1, even though when the indo-1 was added to the cells, the FBSwas capable of temporarily activating the cells.

After the five minute incubation, 40 μl of 100 mM probenecid was addedto the cells to improve loading. The 15 μl of the indo-1 mixture asprepared above was added. The cells were incubated in the dark at 37° C.for 25 minutes.

After the 25 minutes incubation, the cells were centrifuged andresuspended in 10 ml of HBSS and put on ice. One milliliter of the cellsuspension was removed and placed in a FACS machine to measure theresting ratio of violet to green fluorescence from the indo-1. Less than0.1% of the cells exhibited a steady-state fluorescence ratio of lessthan 600. Upon introduction of an appropriate amount of anti-mouse IgMantibody, greater than 58% of the cells exhibited a fluorescence ratioabove 600 after about 100 seconds.

The results for the K46J cells indicated that a B cell can be stimulatedto immobilize calcium by contacting that cell with a ligand that bindsto antibodies on the cell surface.

The above procedure was repeated with the murine B cell line CH27,except that pervanadate was used to stimulate the cells, instead of ananti-IgM antibody. The CH27 exhibited a fluorescence response uponactivation similar to that for the K46J cells. Taken together, theseK46J and CH27 results confirmed the fast response that B cells canconfer on an optoelectronic device incorporating them.

To further illustrate the use of B cells in the devices and methods ofthe invention, M12g3R murine B cells stably expressing an antibodyspecific for the hapten phosphorylcholine were evaluated. These cellsalso contained an emitter molecule aequorin, which was encoded by anexpression plasmid introduced into the cells. About 1×10⁶ of these cellswere removed from their growth medium and resuspended in 1 ml of RPMImedium supplemented with 1% FBS. Twenty microliters of 1 mMcoelenterazine in methanol, supplemented with glutathione was added tothe cells. The mixture was incubated in the dark at room temperature for1 to 4 hours. The solution contained glutathione in order to preventoxidation of the coelenterazine. As discussed above, aequorin oxidizescoelenterazine when calcium levels rise in the cytoplasm, therebyemitting a photon.

After the room temperature incubation, the cells were resuspended in 2ml of HEPES-buffered saline solution supplemented with 10 mM CaCl₂. Thecell density was measured by placing a small drop of the mixture on ahemacytometer. Sixty microliters of cells were then placed in a clearplastic tube, and the tube placed inside a luminometer (i.e., aphotomultiplier tube inside a light-tight box) to measure the backgroundemission rate. The tube was then removed from the luminometer. Twentymicroliters of phosphorylcholine-ovalbumin was then added to the tube,and the tube was placed back into the luminometer for measurement.Within about 50 seconds after adding the phosphorylcholine-ovalbumin,the cells emitted more than 3×10⁵ photons per second, which represents apeak emission rate that was 130 times the background emission rate.

The result from experiment immediately above confirmed that B cellscontaining an emitter molecule and having surface antibodies can bestimulated to emit photons in response to an antigen for which theantibodies are specific. Such cells are useful in the devices andmethods of the invention.

To evaluate whether B cells of non-mammalian origin can be used, threecatfish B cell lines (3B11, IG8, and C4-IE5) were loaded with indo-1,treated, and measured as described above for the CH27 cells. All threecells lines exhibited a substantial fluorescence ratio increase withinabout 100 seconds after adding pervanadate. Thus, B cells fromcold-blooded vertebrates can be used in the devices and methods of theinvention without the temperature controls sometimes required when cellsfrom warm-blooded animals are used.

EXAMPLE 3 Use of Non-B Cells in a Optoelectronic Sensor

To determine if cell types other than B cells were appropriate for usein an optoelectronic sensor, the murine fibroblast cell line BALB/c 3T3was evaluated as follows.

About 7×10⁶ 3T3 cells were removed from a confluent 10 cm² tissueculture plate by trypsinization. These cells were then treated asdescribed above for K46J cells, except that 100 μl of fetal bovine serumwas used to stimulate calcium mobilization instead of an anti-IgMantibody. Before and after stimulation, the fibroblasts exhibited afluorescence ratio similar to that seen for K46J cells as discussedabove, indicating that fibroblasts, as well as B cells, are suitable forthe devices and methods of the invention.

As a further illustration that fibroblasts can be used in place of Bcells in the invention, an in vitro aequorin luminescence assay wasperformed on aequorin produced in 3T3 cells, and the result compared tothat of aequorin produced in K46J cells. The in vitro aequorin assay isdescribed in Button et al., Cell Calcium 14:663-671 (1993).

BALB/c 3T3 cells and K46J cells were transfected with aaequorin-expressing plasmid. Two to three days later, about 7×10⁶ 3T3cells were removed from a confluent 10 cm² tissue culture plate bytrypsinization. A similar number of transfected K46J cells were alsoisolated. Both types of cells were washed with PBS and resuspended in0.5 ml of a solution containing 30 mM Tris (pH 7.6) and 10 mM EDTA. Thecell suspensions were frozen at −80° C. and thawed at room temperaturethree times to release the aequorin from the cells. The cells were thenpelleted by centrifuging for 20 minutes at 4° C. and 14,000 RPM. Thesupernatant containing the aequorin was removed and analyzed.

Fifty microliters of the cell extracts were mixed with 50 μl of reactionbuffer (30 mM Tris [pH 7.6], 10 mM EDTA, and 5% β-mercaptoethanol) and10 μl of 11 μM coelenterazine (Molecular Probes, Inc.). The tubescontaining the mixtures were then wrapped in foil and incubated at roomtemperature for three hours.

At the end of the incubation, one of the tubes was read in a luminometerto obtain a background luminescence level. The 100 μl of a 250 mM CaCl₂solution was added to the extract, and the reading taken again within 30seconds of adding calcium. The procedure was repeated for the othertube. The K46J extract exhibited a 18× increase in photon emission afterthe addition of calcium, while, surprisingly, the 3T3 extract indicateda 23× increase in photon emission after the addition of calcium. Theseresults indicated that aequorin produced in fibroblasts, as well as Bcells, are functional emitter molecules and can be used in the methodsand devices of the invention.

Following the same procedure for the catfish B cells, another non-Bcell, the catfish T cell line 28S.1, was tested for pervanadateactivation. The results indicated that this cold-blooded non-B cell isalso useful in the devices and methods of the invention.

OTHER EMBODIMENTS

All of the features disclosed in this specification may be combined inany combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features.

Furthermore, from the above description, one skilled in the art caneasily ascertain the essential characteristics of the present invention,and without departing from the spirit and scope thereof, can makevarious changes and modifications of the invention to adapt it tovarious usages and conditions. Thus, other embodiments are also withinthe claims.

For example, the device of the invention can further include pumps,valves, fluid reservoirs, batteries, microprocessors, waste removalsystems, and/or temperature control systems.

If desired, different cells binding to different parts of the sampleantigen could be used to look for different identifying features on thesame pathogen. On the one hand, this could be useful in situations wherefalse positives are a problem, since multiple identifying features canbe detected before declaring a positive identification. On the otherhand, it could also be useful where false negatives are a problem, sinceeven if a pathogen lacks one characteristic feature, it could still bedetected on the basis of the other identifying feature.

Analysis of multiple antigens by the device can be performed in parallelfor suitably high concentrations of antigens, and the sample containingthe antigens can be introduced into a chamber containing cells sensitiveto different antigens. However, serial analysis might be necessary forlower concentrations of antigens. The antigen sample can first bedelivered to a cell specific for one antigen, then carried on to adifferent cell specific for another antigen. In one embodiment, a fluidchannel winds back and forth over the surface of the optical detector,presenting the sample to one antigen-specific sector of cells at a time.Flow in the channel can be slow enough or can be periodically stopped inorder to allow antigen-antibody binding. Serial detection is generallyslower than parallel detection, so additional components of the devicecan be added to speed the detection process. Prior information about thesample could be used to reduce the number of antigen-specific types ofcells to which the sample must be presented in series. For example, ifthe sample is known to contain a viral antigen, then it would only haveto be presented to virus-specific cells. Also, the proximal sectors inthe series could be designated for the detection of higher priorityantigens (e.g., more rapidly acting pathogens).

The device can also be designed so that the cell populations can beeasily replaced if they are killed or otherwise rendered ineffective.The support to which the cell lines are attached or in which the mediais contained would be made removable. Extra supports containing the celllines could be frozen or freeze-dried and then stored. After removingold cells from a sensor and inserting fresh cells, one could revive thedevice with the fresh cells.

After the sample has been examined by the cells, it can be flushed away(via the use of a reservoir of fresh medium and a pump, for example)from the cells, thereby preparing the device for the next sample andhelping to prevent contamination or clogging of the device.

1. A device for detecting the presence of an antigen in a sample,comprising: a filter, wherein a sample suspected of comprising anantigen to be detected is passed through said filter; a cell havingantibodies expressed on the surface of the cell that are specific forthe antigen to be detected, wherein binding of the antigen to theantibodies results in an increase in calcium concentration in thecytosol of the cell, the cell further having an emitter molecule which,in response to the increased calcium concentration, emits a photon; aliquid medium in which the cell is immersed, the liquid medium receivingthe sample comprising the antigen to be detected; and an opticaldetector arranged for receiving the photon emitted from the cell.
 2. Thedevice of claim 1, wherein the filter is a passive filter or an activefilter.
 3. The device of claim 1, wherein the cell is a B cell or afibroblast cell.
 4. The device of claim 1, wherein the emitter moleculeis a bioluminescent molecule or a fluorescent molecule.
 5. A device fordetecting the presence of at least one antigen in a sample, comprising:a filter, wherein a sample suspected of comprising at least one antigento be detected is passed through said filter; an array of cells, whereindifferent cells within the array have antibodies expressed on thesurface of the cell that are specific to a different antigen to bedetected, wherein binding of an antigen to the antibodies results in anincrease in calcium concentration in the cytosol of the cell, the cellfurther having an emitter molecule which, in response to the increasedcalcium concentration in the cytosol, emits a photon; a liquid medium inwhich the cell of each sector is immersed, the liquid medium receivingthe sample comprising the antigen to be detected; and an opticaldetector arranged for receiving the photon emitted from the cell.
 6. Thedevice of claim 5, wherein the filter is a passive filter or an activefilter.
 7. The device of claim 5, wherein the cells are B cells,fibroblasts or a combination thereof.
 8. The device of claim 5, whereinthe emitter molecule in each cell is the same or different and areselected from the group consisting of a bioluminescent molecule, afluorescent molecule and a combination thereof.
 9. A method fordetecting the presence of an antigen in a sample, comprising: filteringthe sample suspected of comprising an antigen; introducing the filteredsample into a device, the device comprising a cell immersed in a medium,wherein the cell comprises antibodies expressed on the surface of thecell that are specific for the antigen to be detected, wherein bindingof the antigen to the antibodies results in an increase in calciumconcentration in the cytosol of the cell, the cell further having anemitter molecule which, in response to the increased calciumconcentration, emits a photon; and monitoring photon emission, whereinphoton emission indicates the presence of the antigen in the sample. 10.The method of claim 9, wherein the cell is a cell is a B cell or afibroblast cell.
 11. The method of claim 9, wherein the emitter moleculeis a bioluminescent molecule or a fluorescent molecule.
 12. The methodof claim 9, wherein the antigen is a bacterial antigen or a viralantigen.
 13. A method for detecting the presence of at least one antigenin a sample, comprising: filtering the sample suspected of comprising atleast one antigen; introducing the filtered sample into a device, thedevice comprising an array of cells, wherein different cells within thearray have antibodies expressed on the surface of the cell that arespecific to a different antigen, wherein binding of an antigen to theantibodies results in an increase in calcium concentration in thecytosol of the cell, the cell further having an emitter molecule which,in response to the increased calcium concentration in the cytosol, emitsa photon; and monitoring photon emission from the array of cells,wherein photon emission indicates the presence of at least one antigenin the sample.
 14. The method of claim 13, wherein the cells are Bcells, fibroblasts, or a combination thereof.
 15. The method of claim13, wherein the emitter molecule in each cell is the same or differentand are selected from the group consisting of a bioluminescent molecule,a fluorescent molecule and a combination thereof.
 16. The method ofclaim 13, wherein antigen is a bacterial antigen, a viral antigen, or acombination thereof.